The design of a digital real time autofocusing system of microscope was achieved through this thesis. Three different methods of autofocusing were presented, discussed and designed using MATLAB program. These methods assumed focus functions which indicate the degree of focusing for input image. The method with the high focus value was chosen to be used to calculate the focus measure. To ensure high quality for the output results, the effect of adding noise and image filtering were discussed and simulated using MATLAB. The effect of changing the parameters of each noise type and the type of the filter used was discussed. The system of autofocusing was built and simulated using LABVIEW program supported by the control programs which were designed using MATLAB for autofocusing methods and image filtering. The stepper motor was used in the design to ensure accurate steps for the objective lens of microscope to get the best focal position. The setting of the stepper motor was controlled using LABVIEW program.